title: [Construction and identification of the eukaryotic expression vector expressing short hairpin RNA targeting human COL1A1].
abstract: OBJECTIVE
  To construct the eukaryotic expression vector of short hairpin RNA (shRNA) targeting human collagen type 1 alpha 1 (COL1A1) and observe its inhibiting effect on the expression of target gene.
  
  
  METHODS
  The complementary oligonucleotide sequences coding shRNA were designed and synthesized according to the sequence of human COL1A1 gene, and cloned into the linearized pSilencer(TM);2.1-U6 neo vector. The recombinant vector was confirmed by enzyme digestion analysis and DNA sequencing, and then the positive clones were transfected to human breast cancer cell MDA-MB-231 by Lipofectamine(TM);2000. The stable cell line was selected by G418. The expression of COL1A1 gene was detected by semi-quantitative RT-PCR and Western blot analysis.
  
  
  RESULTS
  Double-enzyme digestion and DNA sequencing verified the correct sequences of the recombinant plasmid pshRNA-COL1A1. Compared with the control group, the expression level of COL1A1 mRNA and protein was inhibited markedly by pshRNA-COL1A1-1 or pshRNA-COL1A1-2 transfection, and the inhibitory rates were respectively (44.41±3.90)%, (63.05±3.13)% in RT-PCR and (45.50±2.71)%, (66.98±2.08)% in Western blot analysis.
  
  
  CONCLUSIONS
  Specific shRNA interference plasmid vector targeting COL1A1 gene mRNA was constructed successfully.
authors.affiliation: Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China.
authors.forename: Hailang
authors.initials: H
authors.lastname: Yu
authors.forename: Jueyu
authors.initials: J
authors.lastname: Zhou
authors.forename: Wei
authors.initials: W
authors.lastname: Meng
authors.forename: Wenling
authors.initials: W
authors.lastname: Zheng
authors.forename: Wenli
authors.initials: W
authors.lastname: Ma
mesh_terms: D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D034741:RNA, Small Interfering